This proposal is intended to charter new directions for design of more selective and more active histone deacetylase (HDAC) inhibitors by mapping the binding "fingerprints" of the HDAC ligands in the different HDAC isoforms using small molecule photoaffinity probes. Since many cancers are associated with aberrant transcriptional activity, and the HDACs can affect transcription factors and gene regulation, these enzymes have been identified as attractive targets for cancer therapy. Indeed, chemical inhibitors of HDACs have been shown to inhibit tumor cell growth and induce differentiation and cell death. At least one non-selective HDAC inhibitor was approved by FDA for cancer and several non-selective HDAC inhibitors are in clinical trials. Multiple studies have shown that therapeutics selective for one or a limited number of specific HDAC isoforms may improve overall efficacy and lower toxicity of these compounds. While some rather limited degree of isoform selectivity has been shown by a few compounds, the problem of identifying selective inhibitors is far from solved. Recent success in the elucidation of the crystal structure of HDAC7, 8 and homologous HDAC proteins has opened a possibility for structure-based drug design. Despite the progress, both ligand-based and structure-based approaches are limited in their applicability to design of isoform selective HDAC inhibitors either because only few isoform selective inhibitors are available as a starting point for ligand based drug design or because high resolution three-dimensional structures are available only for 2 out of 11 class I and II HDAC isoforms. It is also poorly understood how the binding modes of those portions of the HDAC ligands that bind on the surface of the HDAC protein affect the activity of HDAC inhibitors. Clearly, a new approach to this problem is needed. We hypothesize that it will be possible to map the binding modes available to the ligands in different HDAC isoforms by small molecule photoaffinity probes (PAP) and use the resulting knowledge to design isoform selective HDAC inhibitors as potential therapeutics for cancer. Our specific aims in this proposal are as follow: Aim 1: Design and synthesize at least three series of photoaffinity probes (PAPs) consisting of (1) an HDAC ligand, (2) an aromatic azide group for photoaffinity labeling (PAL) of the protein residues directly involved in the binding of the ligand, and (3) an aliphatic azide (a imaging tag attachment group - imTAG) for attaching a fluorescent or a biotin tag. Aim 2: Evaluate the ligands containing either PAL or PAL+imTAG or both groups in photoaffinity labeling experiments with available recombinant purified class I and class II HDACs and determine the modification sites on the proteins using mass-spectrometry proteomics methods. Match the proteomics results with the modeling studies and refine later to better reproduce the experimental findings. Aim 3. Evaluate the cytotoxicity, selectivity, and binding "fingerprints" of the most potent photoaffinity probes in live Hela cells. Aim 4. Incorporate the findings of Aims 1-3 in the structure-based drug design and perform several rounds of CADD, medicinal chemistry, and biological tests to improve the activity and selectivity of the HDAC ligands. PUBLIC HEALTH RELEVANCE: This proposal is intended to charter new directions for design of more selective and more active histone deacetylase (HDAC) inhibitors by mapping the binding "fingerprints" of the HDAC ligands in the different HDAC isoforms using small molecule photoaffinity probes. The ultimate goal of this proposal is to design HDAC inhibitors suitable as HDAC based therapeutics for cancer and other HDAC relevant diseases and conditions.